Functional Analysis of Genes & Proteins: siRNA

ACEA Biosciences xCELLigence

Using the xCELLigence System for Target Identification and Validation

Functional Analysis Fig 1.1 Functional Analysis Fig 1.2

(A) HeLa cells were transfected with various concentrations of KIF11 (the gene encoding Eg5) siRNA.  A time and concentration-dependent antimitotic time-dependent cell response profiles (TCRPs) was observed with higher siRNA concentrations producing more pronounced CI changes. The kinetics of the CI profiles is very similar between various concentrations of siRNAs: CI for the transfected samples started to diverge from the control samples starting 9-12 h post transfection, reaching the lowest level approximately 24 h post transfection, before starting to recover, indicating the specificity of the TCRP.  KIF11 gene express is supported with analysis of Eg5 protein expression by Western blotting (B).  (Data and figures adapted from Ke N, et. al., 2010).

Using “Signature” TCRPs to Identify Compound Properties for Screening

Functional Analysis Fig 2

Compounds with similar mechanisms of action often have similar TCRPs. (A) In A549 cells, both anti-mitotic compounds, paclitaxel and vincristine, mediated the signature anti-mitotic response profile; the DNA damaging agent 5-FU mediated a cytostatic pattern, while the topoisomerase inhibitor camptothecin mediated pronounced cytotoxicity. (B) Cell Index values are consistent with bright-field imaging in the E-Plate View area. (Data and figures adapted from iCELLigence System Application Note No.2, 2012).

Key Benefits:
  1. Real-time monitoring of kinetics of cell responses to treatment can reveal novel information regarding mechanism of action
  2. Assays are performed in tissue culture incubator, allowing for detection of long-term effects
  3. Label-free assay requires no fixation, staining or any other sample processing
  4. Cell response profiles can allow for early identification of unexpected off-target or toxic effects of treatments
Functional Analysis of Genes and Proteins Supporting Information:

  • Functional Analysis of Genes and Proteins Publications:
  1. Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects. Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, Feinstein SC, Wang X, Xu X. Chemistry & biology. 2009 Jul 31;16(7):712-23.
  2. Time-resolved human kinome RNAi screen identifies a network regulating mitotic-events as early regulators of cell proliferation. Zhang JD, Koerner C, Bechtel S, Bender C, Keklikoglou I, Schmidt C, Irsigler A, Ernst U, Sahin O, Wiemann S, Tschulena U. PLoS One. 2011;6(7):e22176. Epub 2011 Jul 13.
  3. Screening and identification of small molecule compounds perturbing mitosis using time- dependent cellular response profiles. Ke N, Xi B, Ye P, Xu W, Zheng M, Mao L, Wu MJ, Zhu J, Wu J, Zhang W, Zhang J, Irelan J, Wang X, Xu X, Abassi YA. Anal Chem. 2010 Aug 1;82(15):6495-503.
  4. RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing’s sarcoma. Arora S, Gonzales IM, Hagelstrom RT, Beaudry C, Choudhary A, Sima C, Tibes R, Mousses S, Azorsa DO. Mol Cancer. 2010 Aug 18;9:218
  5. BH3 response profiles from neuroblastoma mitochondria predict activity of small molecule Bcl-2 family antagonists. Goldsmith KC, Lestini BJ, Gross M, Ip L, Bhumbla A, Zhang X, Zhao H, Liu X, Hogarty MD. Cell Death Differ. 2010 May;17(5):872-82.